Detergent composition containing an alkaline pullulanase from bacillus ferm BP-3048

ABSTRACT

A detergent composition containing an alkaline pullulanase, a surfactant, alkaline agents and/or inorganic electrolytes, divalent metal ion scavengers and bleaching agents is disclosed. The alkaline pullulanase has an optimum pH range of 8.5-10.0 on pullulan, an optimum temperature of about 50° C. and is not deactivated by surfactants. Further, the pullulanase has a strong resistance to almost all detergent components such as chelating agents, proteases, etc. The pullulanase is isolated from Bacillus sp. KSM-AP 1378 deposited as FERM BP-3048. The composition specifically contains 0.1-10 wt. % alkaline pullulanase B, 0.5-60 wt. % surfactant, 0-90 wt. % alkaline agents and/or inorganic electrolytes, 0-50 wt. % divalent metal ion scavengers, and 0-85 wt. % bleaching agents.

This application is a continuation of application Ser. No. 07/681,007,filed on Apr. 5, 1991, now abandoned.

BACKGROUND OF THE INVENTION

1. Field of the Invention:

This invention relates to a detergent composition containing alkalinepullulanase which has excellent detergency especially against starchsoil.

2. Description of the Background Art:

It is a known art that enzymes are compounded into a detergentcomposition. The enzymes in the detergent composition act as awash-aiding agent. They decompose or denature various kinds of soil andstain adhered to clothes, fats and oils, proteins, starch and the likeremained on the dish surface to facilitate removal of the stains.

Heretofore, α-amylase has been used in order to remove starch soils.Detergency against starch soils is improved by soaking the washes in awashing solution containing α-amylase for a reasonably long time.

The inventors of the present invention found that a certain type ofpullulanase effectively acted on starch soils adhered firmly to dishesand fibers, and enabled to improve the detergency remarkably (JapanesePatent Application Laid-Open (Kokai) No. 132192/1990).

However, almost all naturally occurring pullulanases are classified intoneutral or acidic pullulanases which exhibit the maximal and stableenzymatic activity under the neutral or acidic pH conditions. Very fewpullulanases exhibit the maximal activity or alkali-resistance in thealkaline pH range, which are suitable for a detergent composition fordishes or clothes.

An alkaline pullulanase in this invention means a pullulanase having anoptimum pH in the alkaline range. An alkali-resistant pullulanase inthis invention means a pullulanase having an optimum pH in the neutralto acidic range, and exhibiting still sufficient degree of activitieseven in the alkaline range when compared to the activity at its optimumpH while retaining a good stability. The terms `neutral` and `alkaline `are defined as the pH ranges of 6-8 and not less than 8, respectively.

Only alkaline pullulanase known heretofore is that disclosed in JapanesePatent Publication (Kokoku) No. 27786/1978. The above alkalinepullulanase is an enzyme having its optimum pH in the alkaline range andalso having a wider substrate specificity than conventionally knownpullulanases. However, since its optimum pH is in the weak alkalinerange of 8-9, it is not applicable to a detergent component. Inaddition, the above pullulanase has disadvantages that the enzyme isunstable and has low productivity. Therefore, the above pullulanase isnot suitable for the industrial fermentative production.

In view of this situation, the present inventors have carried outextensive studies in order to obtain pullulanases suitable as adetergent component, and, as a result, found that a detergentcomposition having detergency against starch soil was obtained bycompounding an alkaline pullulanase which has its optimum pH in a highpH range and is not deactivated by surfactants. This finding has led tothe completion of the present invention.

SUMMARY OF THE INVENTION

Accordingly, an object of the present invention is to provide a noveldetergent composition comprising an alkaline pullulanase having itsoptimum pH in the alkaline range and being stable against surfactants.

Other objects, features, and advantages of the invention willhereinafter become more apparent from the following description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the relation of reaction pH vs. relative activity of thealkaline pullulanase A used in the present invention.

FIG. 2 shows the reaction of the treatment pH vs. residual activity ofthe alkaline pullulanase A used in the present invention.

FIG. 3 shows the relation of reaction temperature (at pH 9.5) vs.relative activity of the alkaline pullulanase A used in the presentinvention.

FIG. 4 shows the relation of treatment temperature ( at pH 9.5) vs.residual activity of the alkaline pullulanase A used in the presentinvention.

FIG. 5 shows the results of electrophoresis of the alkaline pullulanaseA used in the present invention.

FIG. 6 shows a SDS electrophoresis profile of the alkaline pullulanase Aused in the present invention.

FIG. 7 is a paper chromatography profile showing the production ofmaltooligosaccarides from the enzymatic reaction between the alkalinepullulanase B having an α-amylase activity, and several substrates(pullulan, amylopectin, amylose, and glycogen).

FIGS. 8(a) and 8(b) show the relation of reaction pH vs. relativeactivity of the alkaline pullulanase B having α-amylase activity used inthe present invention.

FIGS. 9(a) and 9(b) show the relation of treatment pH vs. residualactivity of the alkaline pullulanase B having α-amylase activity used inthe present invention.

FIGS.10(a) and 10(b) show the relation of reaction temperature (at pH9.5) vs. relative activity of the alkaline pullulanase B havingα-amylase activity used in the present invention.

FIGS. 11(a) and 11(b) show the relation of treatment temperature (at pH9.5) vs. residual activity of the alkaline pullulanase B used in thepresent invention.

FIG. 12 is an electrophoresis profile of the alkaline pullulanase Bhaving α-amylase activity used in the present invention according to themethod of Davis.

FIG. 13 is a SDS electrophoresis profile of the alkaline pullulanase Bhaving α-amylase activity used in the present invention.

DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS

Alkaline pullulanases used in the present invention are not specificallylimited so far as they have their optimum pH in a high alkaline rangeand are not deactivated by surfactants.

Such alkaline pullulanase include an alkaline pullulanase A and analkaline pullulanase B having the following characteristics:

Alkaline Pullulanase A

1) Action

Decomposes α-1,6 glucoside linkage of pullulan to produce maltotriose.Hydrolyzes α-1,6 glucoside linkage of starch, amylopectin, glycogen, ortheir partial decomposition products.

2) Substrate specificity

Hydrolyzes a branch structure having a degree of polymerization which isnot less than the degree of polymerization of maltose among sugarshaving a branch of α-1,6 glucoside linkage.

3) Working pH and optimum pH range

The working pH is in the range of 5-11 with the optimum pH range of9.5-11.

4) pH stability

Quite stable in the pH range of 8-10, and has relative activity not lessthan 50% even in the pH range of 7-10.5 (treatment: 45° C., 10 minutes).

5) Working temperature and optimum temperature

Acts at a wide temperature range of 10°-60° C. with an optimumtemperature being about 50° C.

6) Thermal stability

Quite stable up to 40° C. when treated in 10 mM glycine-NaCl-NaOH buffersolution (pH 9.5) for 30 minutes.

7) Effects of surfactants

Surfactants such as linear alkylbenzene sulfonate, sodium alkyl sulfate,sodium polyoxyethylene alkylsulfate, sodium α-olefin sulfonate, sodiumα-sulfonated fatty acid ester, sodium alkyl sulfonate, sodium dodecylsulfate, soaps, and Softanol (trade-mark) give almost no adverse effecton activity.

Alkaline Pullulanase B

Has α-amylase activity. In more detail, has the followingcharacteristics:

1) Action

Acts on pullulan and soluble starch to produce mainly maltotriose frompullulan and mainly maltotetraose and maltopentaose from soluble starch.Acts also on glycogen to produce maltotetraose and maltopentaose.

2) Substrate specificity

Acts on pullulan, soluble starch, and glycogen.

3) Working pH and optimum pH

The working pH on pullulan is in the range of 5-12 with the optimum pHin the range of 8.5-10.

The working pH on soluble starch in the range of 4-12 with the optimumph in the range of 7-9.5.

4) pH stability

Stable in the pH range of 6-10.5 against pullulan, and in the pH rangeof 4-12 against soluble starch (treatment: 45° C., 10 minutes).

5) Working temperature and optimum temperature

Acts on pullulan and soluble starch at wide temperature range of 10°-65°C. with an optimum temperature being about 50° C.

6) Thermal stability

Quite stable up to 45° C. when treated in 10 mM glycine-NaCl-NaOH buffersolution (pH 9.5) for 30 minutes.

7) Effects of surfactants

Surfactants such as linear alkylbenzene sulfonate, sodiumpolyoxyethylene alkyl sulfate, sodium α-olefin sulfonate, sodiumα-sulfonated fatty acid ester, sodium alkyl sulfonate, sodium dodecylsulfate, soaps, and Softanol (trade-mark) give almost no adverse effecton activity.

The alkaline pullulanase A used in the present invention is produced,for example, by Bacillus sp. KSM-AP 1876 (FERM BP-3049) which is a kindof alkalophilic microorganism.

This microorganism has the mycological characteristics which will bedescribed below.

The following 21 culture media (Media 1 to 21) were used for theclassification of strains. They all contain 0.5% by weight of sterilizedsodium carbonate (Na₂ CO₃).

Compositions of the culture media used (% by weight)

Medium 1: nutrient broth, 0.8; agar powder (manufactured by Wako PureChemical Co.), 1.5

Medium 2: nutrient broth, 0.8

Medium 3: nutrient broth, 0.8; gelatin 20.0; agar powder (manufacturedby Wako Pure Chemical Co.), 1.5

Medium 4: Bacto litmus milk, 10.5

Medium 5: nutrient broth, 0.8; KNO₃, 0.1

Medium 6: Bacto peptone, 0.7; NaCl, 0.5; glucose, 0.5

Medium 7: SIM agar medium (manufactured by Eiken Kagaku Co.), an amountindicated.

Medium 8: TSI agar medium (manufactured by Eiken Kagaku Co.), an amountindicated.

Medium 9: yeast extract, 0.5; bacto peptone, 1.5; K₂ HPO₄, 0.1; MgSO₄.7H₂ O, 0.02; soluble starch, 2.0; agar powder (manufactured by Wako PureChemical Co.), 1.5

Medium 10: Koser's medium (manufactured by Eiken Kagaku Co.), an amountindicated.

Medium 11: Christensen's medium (manufactured by Eiken Kagaku Co.), anamount indicated.

Medium 12: contains compositions (1) and (2) indicated below to whichare added nitrogen sources consisting of sodium nitrate, sodium nitrite,ammonium chloride, and ammonium phosphate at an amount of 0.25%,0.2025%, 0.158%, and 0.195% by weight respectively in the medium.

(1) yeast extract, 0.05; Na₂ SO₄, 0.1; KH₂ PO₄, 0.1; glucose, 0.1

(2) yeast extract, 0.05; Na₂ SO₄, 0.1; KH₂ PO₄, 0.1; glucose, 1.0;CaCl₂.2H₂ O, 0.05; MnSO₄.4-6H₂ O, 0.01; FeSO₄.7H₂ O, 0.001; MgSO₄.7H₂ O,0.02

Medium 13: King A medium `Eiken` (manufactured by Eiken Kagaku Co.), anamount indicated.

Medium 14: King B medium `Eiken` (manufactured by Eiken Kagaku Co.), anamount indicated.

Medium 15: urea medium `Eiken` (manufactured by Eiken Kagaku Co.), anamount indicated.

Medium 16: cytochrome-oxidase test filter paper (manufactured by NipponPharmaceutical Co.)

Medium 17: 3% aqueous hydrogen peroxide

Medium 18: Bacto peptone, 0.5; yeast extract, 0.5; K₂ HPO₄, 0.1;glucose, 0.1; MgSO₄. 7H₂ O, 0.02

Medium 19: Bacto peptone, 2.7; NaCl, 5.5; K₂ HPO₄, 0.3; glucose, 0.5;bromothymol blue, 0.06; agar powder (manufactured by Wako Pure ChemicalCo.), 1.5

Medium 20: (NH₄)₂ HPO₄, 0.1; KCl, 0.02; MgSO₄. 7H₂ O, 0.02; yeastextract, 0.05; sugar, 1.0

Medium 21: casein, 0.5; yeast extract, 0.5; glucose, 1.0; K₂ HPO₄, 0.1;MgSO₄. 7H₂ O, 0.02; agar powder (manufactured by Wako Pure ChemicalCo.), 1.5

Mycological Characteristics

a) Observation under microscope

Cells are rods of a size of 1.0-2.2 μm×2.2-4.4 μm, with an ellipticalendospore (0.8-1.0 μm×1.0-1.8 μm) forming at their subterminals. Theyhave flagella and are motile. Gram's staining is indefinite. Acidfastness is negative.

b) Growth in various culture media

1) Broth agar plate culture (Medium 1)

Growth of cells is good. Colony has a circular shape, with its surfacebeing smooth and its peripheral end being smooth or wavy. The color ofthe colony is milky semitransparent, and glossy.

2) Broth agar slant culture (Medium 1)

Cells can grow. Colony has a cloth-spreading shape, with a color of thecolony being milky, semitransparent, and glossy.

3) Broth liquid culture (Medium 2)

Cells can grow.

4) Stab culture in broth-gelatin (Medium 3)

growth of cells is good. Liquefaction of gelatin is observed.

5) Litmuth milk medium (Medium 4)

Milk coagulation and peptonization are not observed. Litmuthdiscoloration is indeterminable because the medium is an alkalinemedium.

c) Physiological Characteristics

1) Nitrate reduction and denitrification (Medium 5)

Nitrate reduction is positive.

Denitrification is negative.

2) MR test (Medium 6)

Indeterminable because the medium is an alkaline medium.

3) VP test (Medium 6)

Negative.

4) Production of indole (Medium 7)

Negative.

5) Production of hydrogen sulfide (Medium 8)

Negative.

6) Hydrolysis of starch (Medium 9)

Positive

7) Utilization of citric acid

Negative in Koser's medium (Medium 10), and indeterminable inChristensen's medium (Medium 11).

8) Utilization of inorganic nitrogen sources (Medium 12)

Nitrate, ammonium salts, and nitrate are all utilized.

9) Discoloration (Medium 13, Medium 14)

Negative.

10) Urease (Medium 15)

Negative.

11) Oxidase (Medium 16)

Indeterminable between positive and negative.

12) Catalase (Medium 17)

Positive.

13) Growth range (Medium 18)

Growth temperature: 20°-40° C.

Optimum growth temperature: 30°-35° C.

Growth pH range: 7-10.5

Optimum growth pH: 10

14) Behavior toward oxygen

Aerobic.

15) O-F test (Medium 19)

Discoloration is indeterminable because the medium is an alkalinemedium. Cells can grow only under aerobic conditions.

16) Sugar utilization (Medium 20)

L-Arabinose, D-Xylose, D-glucose, D-mannose, D-fractose, D-galactose,maltose, sucrose, lactose, trehalose, D-sorbitol, D-mannitol, glycerol,starch, salicin, D-ribose and dextrin are utilized.

17) Growth in a medium containing sodium salt (modification of Medium 1)

Cells can grow in the presence of 5% of NaCl, but cannot grow in thepresence of 7% sodium chloride.

18) Hydrolysis of casein (Medium 21)

Positive.

Based on the above mycological characteristics, the strain of thepresent invention was examined referring to Bergey's Mannual ofDeterminative Bacteriology, 8th ed., and `The Genus Bacillus` Ruth, E.Gordon, Agriculture Handbook No. 427, Agricultural Research Service,U.S. Department of Agriculture Washington D.C., (1973), and determinedas an asporogenas rod-shaped microorganism belonging to the genusBacillus. The strain did not grow in the neutral pH range, but grewmostly in the highly alkaline range. From this fact, the strain of thepresent invention was classified as an alkalopholic microorganism whichwas demonstrated by Horikoshi and Akiba [Alkalophilic Microorganism,Japan Scientific Society Press (Tokyo), 1982]. The strain of the presentinvention is thus distinguished from a group of microorganisms belongingto the genus Bacillus which grows in a neutral pH range.

The strain of the present invention has mycologically differentcharacteristics from those of any conventionally known `alkalophilicBaccillus`. Accordingly, the strain of the present invention wasdetermined as a novel strain and named Baccillus sp. KSM-AP 1876, whichwas deposited with Fermentation Research Institute, Agency of IndustrialScience and Technology as FERM BP-3049.

The production of an alkaline pullulanase A used in the presentinvention can be processed by inoculating the above microoganismsaccording to the conventional cultivating methods. It is desirable toadd a suitable amount of carbon and nitrogen sources which themicroorganism can utilize in the medium. There are no specificlimitations as to carbon and nitrogen sources. Exemplary organicnitrogen sources include corn gluten meal, soybean flour, corn steepliquor, casamino acid, yeast extract, pharma media, meat extract,tryptone, soytone, hypro, ajipower, soybean meal, cotton seed meal,cultivator, ajipron, zest. The inorganic nitrogen sources includeammonium sulfate, ammonium nitrate, ammonium phosphate, ammoniumcarbonate, sodium nitrate, ammonium acetate. Exemplary carbon sourcesinclude soluble starch, insoluble starch, amylopectin, glycogen,pullulan, and branched oligomers produced by their partialdecomposition, utilizable carbon sources such as glucose, maltose,arabinose, xylose, ribose, mannose, fructose, galactose, maltose,sucrose, lactose, trehalose, mannitol, sorbitol, glycerol, utilizableorganic acids such as citric acid, acetic acid, and the like. Inaddition to the above carbon and nitrogen sources, inorganic ions suchas phosphate, magnesium, calcium, manganese, zinc, cobalt, sodium,potassium, and the like, and other organic and inorganicmicro-nutritions substances, if necessary, can be added into the culturemedium.

The target alkaline pullulanase A can be collected and purified by meansof conventional collection and purification methods applied to generalenzymes. Specifically, cells can be separated from the culture broth bymeans of conventional solid-liquid separation methods such ascentrifugation, filtration or the like, to obtain a crude enzymesolution. Although it is possible to use the crude enzyme solution thusobtained as it is, it can be served as purified enzyme as required,after separated by means of separation methods such as salting out,precipitation, ultrafiltration and the like to obtain crude enzyme,further purifying and crystallizing the crude enzyme by conventionalmethods.

A preferable method for purifying the alkaline pullulanase A isdiscussed below.

Strains of an alkaline microorganism belonging to genus BacillusKSM-AP1876 are aerobically shake-cultured in a medium containing 1% ofpullulan, 0.2% of tryptone, 0.1% of yeast extract, 0.03% of KH₂ PO₄,0.02% of CaCl₂.2H₂ O, 0.1% of (NH₄)₂ SO₄, 0.001% of FeSO₄.7H₂), 0.0001%of MnCl₂.4H₂ O, 0.02% of MgSO₄.7H₂ O and 0.5% of sodium carbonate, at30° C. for 3 days. Cells are removed from the culture liquid to obtain asupernatant. DEAE-cellulose powder is added to the supernatant to havepullulanase A completely adsorbed onto the DEAE-cellulose. After washingresin with 10 mM tris-HCl buffer solution (pH 8), the enzyme is elutedwith the same buffer solution containing 0.6M NaCl.

Further, after dialyzing and concentrating against 10 Mm tris-HCl buffersolution (pH 8), the enzyme is adsorbed onto DEAE-cellulose DE52equilibrated with the buffer solution. The enzyme is then eluted at aconcentration slant of 0-1M NaCl using the mentioned buffer solution, tocollect active fractions. After concentrating with an ultrafiltrationmembrane whose average fraction-molecular-weight is 10,000, the enzymeis dialyzed using the buffer solution containing 0.1M NaCl overnight.The obtained enzyme is concentrated and dialyzed. Then, after adsorbingonto the Sephacryl S-200 (trade-mark) column equilibrated with the saidbuffer solution containing 0.1M NaCl, the enzyme is eluted with thebuffer solution containing 0.1M NaCl. The active fractions are collectedand adsorbed onto DEAE Toyopearl 650 S (trade-mark) column. The adsorbedenzyme is eluted at a concentration slant of 0.1-1M NaCl in 10 mMtris-HCl buffer solution (pH 8), and the active fractions are collected.The collected active fractions are concentrated using ultrafiltrationmembrane, then adsorbed to butyl Toyopearl 650S (trade-mark) columnequilibrated with the said buffer solution containing 2M ammoniumsulfate. The active fractions are eluted at a concentration slant of2-0M ammonium sulfate using 10 mM tris-HCl buffer solution (pH 8), andcollected. After concentrating with an ultrafiltration membrane, theactive fractions are dialyzed overnight. Purified enzyme thus obtainedgave a single band when subjected to electophoresis using polyacrylamidegel (gel concentration: 15%) and sodium dodecylsulfate (SDS). The activeyield was about 4%.

Enzymological characteristics of the alkaline pullulanase A aredescribed below.

Enzymatic activities were measured using the following buffer solutions(10 mM each) according to the method shown below.

pH 4-6 acetate buffer

pH 6-8 phosphate buffer

pH 8-11 glycine-NaCl-NaOH buffer

pH 11-12 KCl-NaOH buffer

Method for measuring enzymatic activities (pullulanase activities):

0.1 ml of enzyme solution was added to 0.9 ml of a substrate solutionconsisting of a buffer solution and pullulan (final concentration in thereaction system was 0.25%), and the mixture was reacted at 40° C. for 30minutes. After the reaction, reducing sugar was quantitativelydetermined by a method of the 3,5-dinitrosalicylic acid (DNS).

Specifically, 1.0 ml of DNS reagent was added to 1.0 ml of reactionmixture, and the mixture was heated at 100° C. for 5 minutes to developcolor. After cooling, the mixture was diluted by adding 4.0 ml ofdeionized water. This solution was subjected to colorimetricquantitative analysis at a wave length of 535 nm. One unit (1U) ofenzyme activity was defined as the amount of enzyme which released 1μmol of reducing sugar (as glucose) per minute.

Enzymological characteristics

1) Action

Decomposes α-1,6 glucoside linkage of pullulan to produce maltotriose.Hydrolyzes α-1,6 glucoside linkage of starch, amylopectin, glycogen, ortheir partial decomposition products.

2) Substrate specificity

Hydrolyzes a branched structure having a degree of polymerization whichis not less than the degree of polymerization of maltose among sugarshaving a branch at α-1,6 glucoside linkage (Table 1).

                  TABLE 1                                                         ______________________________________                                                                     Relative                                         Substrate     Concentration (M)                                                                            activity (%)                                     ______________________________________                                        Pullulan      1 × 10.sup.-5                                                                          100.0                                            Glycogen (oyster)                                                                           1 × 10.sup.-6                                                                          0.2                                              Glycogen (rabbit liver)                                                                     1 × 10.sup.-6                                                                          0.0                                              Amylopectin   1 × 10.sup.-8                                                                          6.1                                              Amylose       1 × 10.sup.-8                                                                          0.0                                              Maltose       1 × 10.sup.-2                                                                          0.0                                              Maltotriose   1 × 10.sup.-2                                                                          0.0                                              Panose        1 × 10.sup.-2                                                                          0.0                                              Isomaltose    1 × 10.sup.-2                                                                          0.0                                              Isomaltotriose                                                                              1 × 10.sup.-2                                                                          0.0                                              Gentiobiose   1 × 10.sup.-2                                                                          0.0                                              ______________________________________                                    

3) Working pH and optimum pH ranges

The working pH is in the range of 5-11 with optimum pH in the range of9.5-11.

Pullulanase activities at various pHs were measured using reactionsystems consisting of 0.25% of pullulan and each of 10 mM acetate buffer(pH 4-6), phosphate buffer (pH 6-8.5), glycine-NaCl-NaOH buffer (pH8.5-11), and KCl-NaOH buffer (pH 11-12). Each reaction was carried outat 40° C. for 30 minutes. The results are shown in FIG. 1.

4) pH Stability

Quite stable in the pH range of 8-10, and has relative activity not lessthan 50% even in the pH range of 7-10.5.

Pullulanase activities at various pHs were measured using reactionsystems consisting of 0.25% of pullulan and each of 10 mM acetate buffer(pH 4-6), phosphate buffer (pH 6-8.5), glycine-NaCl-NaOH buffer (pH8.5-11), and KCl-NaOH buffer (pH 11-12). Each reaction was carried outat 45° C. for 10 minutes. The results are shown in FIG. 2.

5) Working temperature and optimum temperature

Acts at wide temperature range of 10°-60° C. with an optimum temperaturebeing about 50° C. (FIG. 3).

6) Thermal stability

Temperature at which the present enzyme loses its activity was examinedby subjecting it to heat-treatment at various temperatures at pH 9.5 for30 minutes. It was revealed that the enzyme was stable up to 40° C., andhad residual activity of not less than 50% even at 50° C. (FIG. 4).

7) Molecular Weight

About 120,000±5,000, when measured by means of SDS electrophoresis (gelconcentration: 7.5%) (FIG. 6).

8) Effects of metal ions

Pullulanase activity was adversely affected by 1 mM of Hg²⁺, Cd²⁺, andMn²⁺ strongly, and by 1 mM of Pb²⁺ slightly.

9) Effects of surfactants

Surfactants such as linear alkylbenzene sulfonate, sodium alkyl sulfate,sodium polyoxyethylene alkyl sulfate, sodium α-olefin sulfonate, sodiumα-sulfonated fatty acid ester, sodium alkyl sulfonate, sodium dodecylsulfate, soaps, and Softanol (trade-mark) gave almost no adverse effectswhen the enzyme was treated with 0.05% solution of each of thesurfactants at 40° C. for 15 minutes.

10) Effects of chelating agents

Chelating agents such as EDTA (10 mM), EGTA (10 mM), citric acid (0.05%by weight) gave almost no adverse effects on pullulanase activity.

11) Resistance to protease

The enzyme exhibited strong resistance to any alkaline proteases such asAPI-21 (manufactured by Showa Denko Co.), Maxatase (manufactured by IBISCo.), Sabinase (manufactured by Novo Co.), Alkalase (by Novo Co.),Espelase (by Novo Co.) or the like, when measured in their presence (0.2AU/1).

On the other hand, as for the alkaline pullulanase B usable in thepresent invention, alkaline pullulanase which is produced, for example,by an alkalophilic microroganism such as Bacillus sp KSM-AP 1378 (FERMBP-3048) shown below is mentioned.

This microorganism has the following mycological characteristics.

Mycological Characteristics

a) Observation under microscope

Cells are rods of a size of 0.8-2.4 μm×1.8-4.0 μm, with an ellipticalendospore (1.0-1.2 μm×1.2-1.4 μm) forming at their subterminals. Theyhave flagella and are motile. Gram's strain is indefinite. Acid fastnessis negative.

b) Growth in various culture media

1) Broth agar plate culture (Medium 1)

Growth of cells is good. Colony has a circular shape, with its surfacebeing smooth and its peripheral being smooth. The color of the colony isyellow, semitransparent, and glossy.

2) Broth agar slant culture (Medium 1)

Cells can grow. Colony has a cloth-spreading shape, with color of thecolony being glossy, yellow, and semitransparent.

3) Broth liquid culture (Medium 2)

Cells can grow.

4) Stab culture in broth-gelatin (Medium 3)

Growth of cells is good. Liquefaction of gelatin is observed.

5) Litmuth milk medium (Medium 4)

Milk coagulation and peptonization are not observed. Litmuthdiscoloration is indeterminable because the medium is an alkalinemedium.

c) Physiological characteristics

1) Nitrate reduction and denitrification (Medium 5)

Nitrate reduction is positive. Denitrification is negative.

2) MR test (Medium 6)

Interminable because the medium is an alkaline medium.

3) VP test (Medium 6)

Negative.

4) Production of indole (Medium 7)

Negative.

5) Production of hydrogen sulfide (Medium 8)

Negative.

6) Hydrolysis of starch (Medium 9)

Positive

7) Utilization of citric acid

Negative in Koser's medium (Medium 10), and indeterminable inChristensen's medium (Medium 11).

8) Utilization of inorganic nitrogen sources (Medium 12)

Nitrate, ammonium salts, and nitrite are all utilized.

9) Discoloration (Medium 13, Medium 14)

Negative.

10) Urease (Medium 15)

Negative.

11) Oxidase (Medium 16)

Negative

12) Catalase (Medium 17)

Positive

13) Growth range (Medium 18)

Growth temperature: 20°-40° C.

Optimum growth temperature: 30°-35° C.

Growth pH range: 7-10.5

Optimum growth pH: 10

14) Behavior towards oxygen

Aerobic.

15) O-F test (Medium 19)

Discoloration is indeterminable because the medium is an alkalinemedium. Cells can grow only under aerobic conditions.

16) Sugar utilization (Medium 20)

L-Arabinose, D-xylose, D-glucose, D-mannose, D-fractose, D-galactose,maltose, sucrose, lactose, trehalose, D-sorbitol, D-mannitol, inositol,glycerol, starch, raffinose, salicin, D-ribose, and dextrin areutilized.

17) Growth in a medium containing sodium salt (modification of Medium 1)

Cells can grow in the presence of 7% NaCl but cannot grow in thepresence of 10% NaCl

18) Hydrolysis of casein (Medium 21)

Positive

Based on the above mycological characteristics, the strain of thepresent invention was examined referring to Bergey's Mannual ofDeterminative Bacteriology, 8th ed., and `The Genus Bacillus` Ruth, E.Gordon, Agriculture Handbook No. 427, Agricultural Research Service,U.S. Department of Agriculture, Washington D.C. (1973), and determinedas an asporogenas rod-shaped microorganism belonging to the genusBacillus. The strain did not grow in a neutral pH range, but grew mostlyin a highly alkaline range. From this fact, the strain of the presentinvention was classified as an alkalophilic microorganism which wasdemonstrated by Horikoshi and Akiba [Alkalophilic Microorganisms, JapanScientific Society Press (Tokyo), 1982]. The strain of the presentinvention is distinguished from a group of microorganisms belonging tothe genus Bacillus which grows in a neutral pH range.

The strain of the present invention has mycologically differentcharacteristics from those of any conventionally known `alkanophilicBacillus`. Accordingly, the strain of the present invention wasdetermined as a novel strain, and named Bacillus sp. KSM-AP 1378, whichwas deposited with Fermentation Research Institute, Agency of IndustrialScience and Technology as FERM BP-3048.

The production of the alkaline pullulanase B used in the presentinvention can be processed by inoculating the above microorganismsaccording to the conventional cultivating methods. It is desirable toadd a suitable amount of carbon and nitrogen sources which themicroorganism can utilize in the medium. There are no specificlimitations as to carbon and nitrogen sources.

Exemplary nitrogen sources include corn gluten meal, soybean flour, cornsteep liquor, casamino acid, yeast extract, pharma media, meat extract,tryptone, soytone, hypro, ajipower, cotton seed meal, cultivator,ajipron and zest. The inorganic nitrogen sources include ammoniumsulfate, ammonium nitrate, ammonium phosphate, ammonium carbonate,sodium nitrate, ammonium acetate and the like. Exemplary carbon sourcesinclude soluble starch, insoluble starch, amylopectin, glycogen,pullulan and branched oligomers produced by their partial decomposition,and utilizable carbon sources such as glucose, maltose, arabinose,xylose, ribose, mannose, fructose, glactose, sucrose, lactose,trehalose, mannitol, sorbitol, glycerol, and utilizable organic acidssuch as citric acid, acetic acid and the like. In addition to the abovecarbon and nitrogen sources, inorganic ions such as phosphate,magnesium, calcium, manganese, zinc, cobalt, sodium, potassium and thelike, and other organic or inorganic micronutritious substances, ifnecessary, can be added into the culture medium.

A preferable culturing temperature is from 20° to 40° C., mostpreferably from 30° to 35° C., and pH is from 8 to 10.5, preferably 10.The cultivation is usually completed in 2-3 days under the aboveconditions.

The target alkaline pullulanase B having α-amylase activity can becollected and purified by means of conventional collection andpurification methods applied to general enzymes. Specifically, cells canbe separated from the culture broth by means of conventionalsolid-liquid separation methods such as centrifugation, filtration orthe like, to obtain a crude enzyme solution.

Although it is possible to use the crude enzyme solution thus obtainedas it is, it can be served as purified enzyme, after separated by meansof separation methods such as salting out, precipitation,ultrafiltration and the like to obtain a crude enzyme, further purifyingand crystalyzing the crude enzyme by conventional methods, as required.

A preferable method for purifying alkaline pullulanase B is discussedbelow.

Strains of an alkaline microorganism belonging to genus Bacillus KSM-AP1378 are aerobically shake-cultured in a medium containing 1% ofpullulan, 1% polypeptone, 0.5% of yeast extract, 0.1% of KH₂ PO₄, 0.25%of Na₂ HPO₄.12H₂ O, 0.02% of MgSO₄.7H₂ O and 0.5% of sodium carbonate,at 30° C. for 3 days. Cells are removed from the culture broth to obtaina supernatant. The supernatant is then purified by means of DEAEcellulose adsorption, α-cylodextrin affinity chromatography, DEAEToyopearl (manufactured by Toyo Soda Co.) chromatography, and Sephacryl(manufactured by Pharmacia Co.) chromatography. Purified enzyme thusobtained gave a single band when it is subjected to electophoreses usingpolyacrylamide gel (gel concentration: 15%) or sodium dodecyl sulfate(SDS). In this process, the active yield was about 2%.

Alkaline pullulanase B thus obtained is preferably used as an componentof the detergent composition of the present invention. Enzymologicalcharacteristics of alkaline pullulanase B are explained below.

Enzymatic activities were measured using the following buffer solutions(50 mM each) according to the method explained below.

pH 4-6: acetate buffer

pH 6-8: phosphate buffer

(for measuring pullulanase activity)

pH 6-8: Tris-malate buffer

(for measuring α-amylase activity)

pH 8-11: glycine-NaCl-NaOH buffer

pH 11-12: KCl-NaOH buffer

Method for measuring enzymatic activities:

1) Pullulanase activity

Same procedures were followed as mentioned in the section of alkalinepullulanase A.

2) α-amylase activity

0.1 ml of enzyme solution was added to 0.9 ml of a substrate solutionconsisting of a buffer solution and soluble starch (final concentrationin the reaction system was 0.25%), and the mixture was reacted at 50° C.for 15 minutes. After the reaction, reducing sugar was quantitativelydetermined by means of a DNS method. Specifically, 1.0 ml of DNS reagentwas added to 1.0 ml of reaction mixture, and the mixture was heated at100° C. for 5 minutes to develop color. After cooling, the mixture wasdiluted by adding 4.0 ml of deionized water. This solution was subjectedto colorimetric quantitative analysis at wave length of 535 nm. One unit(1U) of enzyme activity was defined as the amount of enzyme whichreleased 1 μmol of reducing sugar (as glucose) per minute.

Enzymological Characteristics

1) Action

Acts on pullulan and soluble starch to produce mainly maltotriose frompullulan and mainly maltotetraose and maltopentaose from soluble starch.Acts also on glycogen to produce maltotetraose and maltopentaose (FIG.7).

2) Substrate specificity

Acts on pullulan, soluble starch, and glycogen (Table 2).

                  TABLE 2                                                         ______________________________________                                                                     Relative                                         Substrate     Concentration (M)                                                                            activity (%)                                     ______________________________________                                        Pullulan       0.25%         100                                              Glycogen (oyster)                                                                           0.5%           25                                               Glycogen (rabbit liver)                                                                     0.5%           24                                               Amylopectin   0.5%           55                                               Amylose       0.5%           28                                               Maltose       1 × 10.sup.-2 M                                                                         0                                               Maltotriose   1 × 10.sup.-2 M                                                                         0                                               Panose        1 × 10.sup.-2 M                                                                         0                                               Isomaltose    1 × 10.sup.-2 M                                                                         4                                               Isomaltotriose                                                                              1 × 10.sup.-2 M                                                                        11                                               Gentiobiose   1 × 10.sup.-2 M                                                                         0                                               ______________________________________                                    

3) Working pH and optimum pH ranges

The working pH on pullulan of alkaline pullulanase B is in the range of5-12 with an optimum pH in the range of 8.5-10.

Pullulanase activities at various pHs were measured using reactionsystems consisting of 0.25% of pullulan and each of 10 mM acetate buffer(pH 4-5), phosphate buffer (pH 6-8), glycine-NaCl-NaOH buffer (pH9-10.5), and KCl-NaOH buffer (pH 11-12). Each reaction was carried outat 40° C. for 30 minutes. The results are shown in FIG. 8(a).

The working pH on soluble starch was in the range of 4-12 with anoptimum pH in the range of 7-9.5. α-Amylase activities at various pHswere measured using reaction systems consisting of 0.25% of solublestarch and each of 10 mM acetate buffer (pH 4-5), tris-malate buffer (pH6-8), glycine-NaCl-NaOH buffer (pH 9-10.5), and KCl-NaOH buffer (pH11-12). Each reaction was carried out at 50° C. for 15 minutes. Theresults are shown in FIG. 8(b).

4) pH stability

Alkaline pullulanase B is stable in the pH range of 6-10.5 againstpullulan. Pullulanase activities at various pHs were measured usingreaction systems consisting of 0.25% of pullulan and each of 10 mMacetate buffer (pH 4-5), phosphate buffer (pH 6-8), glycine-NaCl-NaOHbuffer (pH 9-10.5), and KCl-NaOH buffer (pH 11-12). Each reaction wascarried out at 45° C. for 10 minutes. The results are shown in FIG.9(a).

Alkaline pullulanase B of this invention was stable in the pH range of4-12 against soluble starch. α-Amylase activities at various pHs weremeasured using reaction systems consisting of 0.25% of pullulan and eachof 10 mM acetate buffer (pH 4-5), tris-malate buffer (pH 6-8),glycine-NaCl-NaOH buffer (pH 9-10.5), and carbonate buffer (pH 11-12).Each reaction was carried out at 50° C. for 15 minutes. The results areshown in FIG. 9(b).

5) Working temperature and optimum temperature

Acts on pullulan and soluble starch at a wide temperature range of10°-65° C. with an optimum temperature being about 50° C. The resultsare shown in FIGS. 10(a) and 10(b).

6) Thermal stability

Temperature at which the present enzyme loses its activity was examinedby subjecting it to heat-treatment at various temperatures at pH 9.5 for30 minutes. As a result, the enzyme was very stable up to 45° C. Theresults are shown in FIGS. 11(a) and (b).

7) Molecular weight

About 200,000±5,000, when measured by means of SDS electrophoresis (gelconcentration: 7.5%). (FIG. 13)

8) Effects of metal ions

Pullulanase activity was adversely affected by Hg²⁺, Mn²⁺, and Pb²⁺ions, and α-amylase activity was adversely affected by Hg²⁺, Mn²⁺, Pb²⁺,Cd²⁺, and Zn²⁺. The results are shown in Table 3.

                  TABLE 3                                                         ______________________________________                                                         Residual activity (%)                                                               Pullulanase                                                                              α-Amylase                             Compound  Concentration                                                                              activity   activity                                    ______________________________________                                        No addition                                                                             --           100        100                                         Metal salt                                                                    HgCl.sub.2                                                                               1 mM        36         53                                          MnCl.sub.2                                                                               1 mM        51         28                                          PbCl.sub.2                                                                               1 mM        53         53                                          CdCl.sub.2                                                                               1 mM        80         38                                          ZnCl.sub.2                                                                               1 mM        93         57                                          Chlating agent                                                                EDTA      10 mM        77         17 (88)*                                    EGTA      10 mM        65         18 (91)*                                    ______________________________________                                         *The values in parentheses are residual activities when Ca.sup.2+ ions        were added again.                                                        

As shown in Table 3, the metal ions which adversely affect thepullulanase activity and those which adversely affect the α-amylaseactivity are discriminated from each other.

9) Effects of surfactants

0.05% Solution of surfactants such as linear alkylbenzene sulfonate,sodium polyoxyethylene alkylsulfate, sodium α-olefin sulfonate, sodiumα-sulfonated fatty acid ester, sodium alkyl sulfonate, sodium dodecylsulfate, soaps, and softanol were tested at 40° C. for 15 minutes togive almost no adverse effect on enzyme activities.

10) Effects of chelating agents

Chelating agents such as EDTA (10 mM), EGTA (10 mM) gave almost noadverse effect on pullulanase activity. But α-amylase activity wasadversely affected significantly. The α-amylase activity which had beenadversely affected by chelating agents can be restored by the additionof Ca²⁺ ions as shown in Table 3.

11) Resistance to protease

In the presence of an alkaline protease, for example, Maxatase(manufactured by IBIS Co.), Sabinase (manufactured by Novo Co.) or thelike in an amount of 0.2 AU/1, the activities were measured, to confirmthat the enzyme of this invention exhibited strong resistance to anyproteases.

As is apparent from the above-mentioned enzymological characteristics,the alkaline pullulanase B of the present invention is the one havingdifferent physiological and chemical characteristics from conventionalpullulanase having α-amylase activity.

Further, in order to clarify the novelty of the enzyme of the presentinvention, comparison data on physiological and chemical characteristicsof the enzyme of the present invention and conventional pullulanaseshaving α-amylase activity are shown in Table 4.

As is apparent from Table 4, the alkaline pullulanase B of the presentinvention is clearly different from a pullulanase-amylase complexderived from Bacillus subtilis TU or from an amylase having pullulanaseactivity derived from Bacillus circulans F-2.

                                      TABLE 4                                     __________________________________________________________________________                Pullulanase                                                                   Japanese Patent                                                                       Japanese Patent                                                                          Present                                                    Publication                                                                           Laid-open  Invention                                                  (Kokoku) No.                                                                          (Kokai) No.                                                                              (Alkaline                                                  18717/1989                                                                            60376/1989 Pullulanase B)                                 __________________________________________________________________________    Microorganism                                                                             Bacillus subtilis                                                                     Bacillus circulans                                                                       Bacillus sp.                                               Tu      F-2        KSM-AP 1378                                    Optimum of pH of                                                                          7.0-7.5 7.0         8.5-10.0                                      pullulanase activity                                                          Optimum pH of                                                                             6.7-7.0 7.0-8.5    7.0-9.5                                        α-amylase activity                                                      Optimum temperature of                                                                    50° C.                                                                         50° C.                                                                            50-55° C.                               pullulanase activity                                                          Optimum temperature of                                                                    60° C.                                                                         50° C.                                                                            45-50° C.                               α-amylase activity                                                      Molecular weight                                                                          450,000 218,000    200,000                                                    (gel filtration)                                                                      (SDS electrophoresis)                                                                    (SDS electrophoresis)                          Major product from                                                                        G1, G2, G3                                                                            G4, G5, G6 G4, G5                                         soluble starch*                                                               Major products from                                                                       G3      G3         G3                                             pullulan*                                                                     __________________________________________________________________________     *: G1: glucose, G2: maltose, G3: maltotriose, G4: maltotetraose, G5:          maltopentaose, G6: maltohexaose                                          

The above-mentioned alkaline pullulanase A or alkaline pullulanase B aregenerally incorporated into a detergent composition of the presentinvention by 0.1-10 wt. %. These alkaline pullulanases can be usedeither in the purified form of enzyme, or as an crude enzyme which isculture broth itself.

Other conventional detergent components may optionally be compoundedinto the detergent composition of the present invention depending on theuse and the purposes. These components to be compounded are illustratedbelow.

1) The amount of surfactants is not limited especially, but ispreferably 0.5-60% by weight. The exemplary surfactants usable for thedetergent comosition of the present invention include nonionicsurfactants such as alkylbenzene sulfonate, alkyl or alkenylethersulfate, alkyl or alkenyl sulfate, olefin sulfonate, alkane sulfonate,saturated or unsaturated fatty acid salt, alkyl or alkenyl ethercarboxylic acid salt, α-sulfo fatty acid salt or ester, aminoacid-typesurfactant, N-acylamino acid-type surfactant, alkyl or alkenylacid-phosphate, alkyl or alkenyl phosphate or its salt; amphotericsurfactants such as carboxyl or sulfobetaine-type surfactant; nonionicsurfactants such as polyoxyalkylene alkyl or alkenyl ether,polyoxyethylene alkyl phenyl ether, higher fatty acid alkanolamide orits alkylene oxide adduct, sucrose fatty acid ester, fatty acid glycerolmomoester, alkylamine oxide, alkylglycoside; and cationic surfactantssuch as quaternary ammonium salt.

When the detergent composition of this invention is used for automaticdish-washing, little- or non-foaming nonionic surfactants arepreferable. As for these kinds of surfactants, alkoxylated nonionicsurfactants (alkoxyl moiety is selected from the group consisting ofethylene oxide, propylene oxide and their mixtures) are enumerated.Mention may be given to `Plurafac LF 403` and `Plurafac LF 1300` (bothtrade-marks, manufactured by BASF Japan Co.), `Softanol EP 7045`(trade-mark, manufactured by Nippon Shokubai Kagaku Kogyo K. K.). Whenthe detergent composition is used for an automatic dish-washer, it isperferable that the surfactants of 0.5%-30% by weight are compoundedinto the composition.

2) Alkaline agents such as carbonate, hydrogencarbonate, silicate,boronate and alkanolamine and inorganic electrolytes such as sulfate areusually compounded into the composition in an amount of 0-90% by weight.

3) Phosphate such as tri-poly phosphate, pyrophosphate, orthophosphate;phosphonates such as ethane-1, 1-diphosphonate; phosphono carboxylatessuch as 2-phosphonobutane-1,2-dicarboxylate; amino acid salts such asaspartate, glutamate; aminopolyacetates such as nitrilo triacetate,ethylenediamine tetraacetate; polymer-chelating agents such aspolyacrylate, polyakonitate; organic acid salts such as oxalate,citrate; divalent metal ion scavengers such as alumino silicate areusually compounded into the composition in an amount of 0-50% by weight.

4) Bleaching agents such as sodium percabonate, sodium perboronate,sodium hypochloride, dichloro isosialic acid are compounded into thecomposition in an amount of 0-85% by weight.

5) As for the other small quantity components which may be incorporatedas required, mention may be given to anti-recontamination agents such aspolyethylene glycol, carboxy methyl cellulose; enzymes such as protease,lipase, α-amylase, cellulase; anti-enzyme quenchers such as sulfite;fluorescent dyes; blueing agents; colorants; anti-caking agents;solubilizing agents; activating agents for enzymes or bleaching agents;anti-metal-corrosion agents.

Exemplary proteases to be used in the present invention includesubtilicins obtained from specific strains such as Bacillus subtilis andBacillus licheniformis. For example, `Maxatase` (trade-mark,Gist-Brocades), `Alkalase`, `Esperase` and `Sabinase` (trade-marks, NovoIndustry Co.), are enumerated.

α-Amylase to be used in the present invention are obtained, for example,from Bacillus licheniformis or Bacillus subtilis. Commercially availableexamples are `Termamyl` (trade-mark, Novo Industry Co.), `Maxamyl`(trade-mark, Gist-Brocades).

When the detergent composition is used for an automatic dish-washer ofpower or granule forms, inorganic alkaline agents are preferably used asthe balance components. Such inorganic alkaline agents include sodiumpyrolate, sodium orthophosphate, sodium tripolyphosphate, sodiumcarbonate, sodium hydrogencarbonate, sodium sesquicarbonate, borax,sodium silicate and the like. Since sodium silicate works as ananti-metal-corrosion agent, it is desirable to use it together withother alkaline agents. It is the most desirable that the sodium silicate(SiO₂ /Na₂ O=1/1-4/1, preferably 2/1-2.5/1) (concentration: 2-15% byweight) and the other alkaline agents (concentration: 35-85% by weight)are used together. The amount of the inorganic alkaline agents arecontrolled so that the pH of a detergent solution (concentration:0.05-1% by weight) falls in the range of is 9.0-11.0. When the detergentcomposition is liquid, balance component is water.

Fatty acids, hydrocarbon-chain-length of which is in a degree of 8-18,benzotriazole and the like are effectively added to the detergentcomposition for an automatic dishwasher as an anti-copper-corrosionagent.

Recently, environmental problems caused from phosphate-containingdetergents has become a controvercial subject. Accordingly, it isimportant that the detergents should contain no phosphor whilemaintaining good detergency against various kinds of contamination.

When removing phosphor, hydroxy polyhydric carboxyl acids or their watersoluble salts of formula (I) are preferably used as divalent metal ionscavengers. ##STR1## wherein X represents --H, --CH₃, --CH₂ COOH or--CH(OH)COOH and Y represents --H or --OH.

Among the above compounds, citric acid, malic acid, tartaric acid ortheir water soluble salts are preferably used. As for the salts, saltsof sodium, potassium, monoethanolamine, diethanolamine, triethanolamineand the like are mentioned.

Such components are preferably compounded into the detergent compositionof the present invention in an amount of 0.5-30% by weight.

Further, polymer chelating agents in an amount of 1-10% by weight arepreferably used as divalent-metal scavengers. The illustrative polymerchelating agents include those described Japanese Patent ApplicationLaid-open (Kokai) No. 145199/1982, and polymers of acrylic ormethacrylic acid, acrylic acid-methacrylic acid copolymer, their watersoluble salts and the like. Their average molecular weight is1,500-100,000, preferably 3,000-20,000.

The detergent compositions of the present invention are suitably used asdetergents for clothings, dishes, households and the like in the form ofpowder, granule, or liquid, by blending the above-mentioned componentsaccording to conventional methods.

Other features of the invention with becomes apparent in the course ofthe following description of the exemplary embodiments which are givenfor illustration of the invention and are not intended to limit thereof.

EXAMPLES PREPARATION EXAMPLES 1

A spoonful of soil (about 0.5 g) of Yokohama-city, Kanagawa-prefecture,Japan was suspended in sterilized saline, and the mixtures washeat-treated at 80° C. for 15 minutes. Supernatant of the heat-treatedmixture was appropriately diluted, and applied onto an agar medium(Medium A) for separating, then cultivating at 30° C. for 3 days to growcolonies. The colonies which formed transparent zones in theirperipheries due to pullulan dissolution were collected to obtain strainswhich produce pullulanase A. Further, these strains were inoculated intothe liquid medium (Medium B), and shake-cultivated at 30° C. for 3 days.

After the cultivation, the culture broth was centrifuged to separate asupernatant. The pullulanase activities were measured at pH 10 to selectstrains which produce alkaline pululanase A. Bacillus sp. KSM-AP 1876(FERM BP-3049) which is a microorganism capable of producing alkalinepullulanase A was thus obtained.

    ______________________________________                                        Medium A     Pullulan     0.8%                                                             Colored pullulan                                                                           0.2                                                              Polypeptone  0.2                                                              Yeast extract                                                                              0.1                                                              KH.sub.2 PO.sub.4                                                                          0.03                                                             (NH.sub.4).sub.2 SO.sub.4                                                                  0.1                                                              MgSO.sub.4.7H.sub.2 O                                                                      0.02                                                             CaCl.sub.2.2H.sub.2 O                                                                      0.02                                                             FeSO.sub.4.7H.sub.2 O                                                                      0.001                                                            MuCl.sub.2.4H.sub.2 O                                                                      0.0001                                                           Agar         1.5                                                              Na.sub.2 CO.sub.3                                                                          0.5                                                 Medium B     pH 10.0                                                                       Pullulan     1.0%                                                             Tryptone     0.2                                                              Yeast extract                                                                              0.1                                                              KH.sub.2 PO.sub.4                                                                          0.03                                                             (NH.sub.4).sub.2 SO.sub.4                                                                  0.1                                                              MgSO.sub.4.7H.sub.2 O                                                                      0.02                                                             CaCl.sub.2.2H.sub.2 O                                                                      0.02                                                             FeSO.sub.4.7H.sub.2 O                                                                      0.001                                                            MnCl.sub.2.4H.sub.2 O                                                                      0.0001                                                           Na.sub.2 CO.sub.3                                                                          0.5                                                 ______________________________________                                    

2) The strains of Bacillus sp. KMS-AP 1876 which produce alkalinepullulanase A were inoculated into the liquid Medium B, andshake-cultivated at 30° C. for 3 days. After the cultivation, cells wereremoved by means of centrifugation to obtain a crude pullulanasesolution. The solution was processed according to a conventional methodto prepare ethanol-dried powder. The crude enzyme samples shown in Table5 were obtained. Enzymatic activities were measured at pH 9.

                  TABLE 5                                                         ______________________________________                                                   Amount of crude enzyme                                                                          Enzyme                                           Strain     per 1 l of medium (g)                                                                           activity (U/g)                                   ______________________________________                                        KSM-AP 1876                                                                              0.2               1870                                             ______________________________________                                    

3) The crude enzyme solution prepared in 2) was treated by the followingsteps 1-5.

1. DEAE-cellulose adsorption

2. DEAE-cellulose (manufactured by Wattman Co.) Chromatography

3. Sephacryl (manufactured by Pharmacia Co.) Chromatography

4. DEAE Toyopearl (manufactured by Toyo Soda Co.) Chromatography

5. Butyl Toyopearl (manufactured by Toyo Soda Co.) Chromatography

The crude enzyme was thus purified to obtain alkaline pullulanase A.

The alkaline pullulanase A obtained was subjected to electrophoresisaccording to the method of Davis [Davis D. J., Ann. N.Y. Acad. Sci.,121, 404, (1964)], and stained with Coomassie brilliant Blue to confirmthat it gave a single band (FIG. 5).

4) The alkaline pullulanase A obtained in 3) was subjected to sodiumdodecyl sulfate (SDS) electrophoresis according to a conventional methodto confirm that the enzyme had a molecular weight of 120,000±5,000.

PREPARATION EXAMPLE 2

A spoonful of soil (about 0.5 g) of Tochigi-city, Tochigi-prefecture,Japan was suspended in sterilized saline, and the mixture washeat-treated at 80° C. for 15 minutes. Supernatant of the heat-treatedmixture was appropriately diluted, and applied onto an agar medium(Medium C) for separation, then cultivated at 30° C. for 3 days to growcolonies. The colonies which formed transparent zone in theirperipheries due to dissolution of colored pullulan and colored starchwere collected to obtain strains which produce pullulanase B. Further,these strains were inoculated into the liquid medium (Medium D), andshake-cultivated at 30° C. for 3 days. After the cultivation, theculture broth was centrifuged to separate a supernatant. The pullulanaseactivities were measured at pH 10 to select strains which producealkaline pullulanase B. Bacillus sp. KSM-AP 1378 (FERM BP-3048) which isa microorganism capable of producing alkaline pullulanase B was thusobtained.

    ______________________________________                                        Medium C     Pullulan     0.5%                                                             Soluble Starch                                                                             0.5                                                              Colored Pullulan                                                                           0.2                                                              Colored Starch                                                                             0.2                                                              Polypeptide  0.2                                                              Yeast extract                                                                              0.1                                                              KH.sub.2 PO.sub.4                                                                          0.03                                                             (NH.sub.4).sub.2 SO.sub.4                                                                  0.1                                                              MgSO.sub.4.7H.sub.2 O                                                                      0.02                                                             CaCl.sub.2.2H.sub.2 O                                                                      0.02                                                             FeSO.sub.4.7H.sub.2 O                                                                      0.001                                                            MnCl.sub.2.4H.sub.2 O                                                                      0.0001                                                           Agar         1.5                                                              Na.sub.2 CO.sub.3                                                                          0.5                                                 Medium D     pH 10.0                                                                       Pullulan     0.5%                                                             Soluble Starch                                                                             0.5                                                              Tryptone     0.2                                                              Yeast extract                                                                              0.1                                                              KH.sub.2 PO.sub.4                                                                          0.03                                                             (NH.sub.4).sub.2 SO.sub.4                                                                  0.1                                                              MgSO.sub.4.7H.sub.2 O                                                                      0.02                                                             CaCl.sub.2.2H.sub.2 O                                                                      0.02                                                             FeSO.sub.4.7H.sub.2 O                                                                      0.001                                                            MnCl.sub.2.4H.sub.2 O                                                                      0.0001                                                           Na.sub.2 CO.sub.3                                                                          0.5                                                 ______________________________________                                    

2) The strains of Bacillus sp. KSM-AP 1378 which produce alkalinepullulanase B having α-amylase activity were inoculated into the liquidMedium D, and shake-cultivated at 30° C. for 3 days. After thecultivation, cells were centrifugally separated to obtain a crude enzymesolution, which was processed according to a conventional method toprepare ethanol-dried power. A crude enzyme sample shown in Table 6 wasobtained. Enzymatic activities were measured at pH 9.

                  TABLE 6                                                         ______________________________________                                                Amount of crude                                                                          Enzyme                                                             enzyme per 1 l of                                                                        activity (U/g)                                             Strain    medium (g)   Pullulanase                                                                              α-amylase                             ______________________________________                                        KSM-AP 1378                                                                             0.2          2096       2476                                        ______________________________________                                    

3) To a supernatant of the crude enzyme prepared in 2) was addedDEAE-cellulose powder, and pullulanase in the supernatant was completelyadsorbed onto the DEAE-cellulose. After the resin was washed with 10 mMtris-HCl buffer solution (pH 8), the enzyme was eluted with 10 mMtris-HCl buffer solution (pH 8) containing 0.6M NaCl. The eluate wasdialyzed against 10 mM tris-HCl buffer solution (pH 8). Then, the enzymewas adsorbed onto an α-cyclodextrin affinity column equilibrated with 10mM tris-HCl buffer solution, and eluted with 10 mM tris-HCl buffersolution (pH 8) containing β-cyclodextrin to collect active fractions.After dialyzing, the active fractions were adsorbed to DEAE Toyoperearl650 S (trade-mark) equilibrated with 10 mM tris-HCl buffer solution (pH8). The enzyme adsorbed was gradiently eluted with 10 mM tris-HCl buffercontaining NaCl having concentration of 0.1-1M to collect activefractions. After dialyzing, the active fractions were filled into aSephacryl S-200 column equilibrated with 10 mM tris-HCl buffer solution(pH 8) containing 0.1M NaCl and eluted with the same buffer containing0.1M NaCl to collect active fractions. The collected active fractionswere concentrated on an ultrafiltration membrane and dialyzed overnightagainst 10 mM tris-HCl buffer solution (pH 8) to obtain alkalinepullulanase B having α-amylase activity. The obtained alkalinepullulanase B was subjected to electrophoresis according to the methodof Davis [Davis D. J., Ann. N.Y. Acad. Sci., 121, 404, (1964)], andstained with Coomassie Brilliant Blue to confirm that it gave a singleband (FIG. 12).

5) The alkaline pullulanase B having α-amylase activity obtained in 4)was subjected to sodium dodecyl sulfate (SDS) electrophoresis accordingto a conventional method. The results are shown in FIG. 13. It wasconfirmed that the enzyme had a molecular weight of 200,000±5,000.

EXAMPLE 1 Detergent for automatic dish-washer

Washing conditions, detergency tests, and the results were as follows.

1) Washing conditions

Washing machine used: All-automatic dish-washer (NP-600, manufactured byMatsushita Electrical Industry Co.). This machine is of the type that adetergent solution is jet-sprayed from a rotary nozzle and washes thedishes arranged above the jet orbit of the detergent solution.

Washing temperature: Temperature was raised slowly from 5° C. to 55° C.

Water for washing: Water hardness=3.5° DH

Detergent concentration: 0.2%

Washing time:

Washing; 20 minutes,

Rinsing; 20 minutes.

Amount of circulated water during washing: 2.5 l

2) Evaluation of detergency

Dishes contaminated by starch soil and the evaluation method:

Contaminated dishes

Rice flour and boiled rice grains were mixed (9:1), and further mixedwith a mixer after adding an equal amount of tap water. The above soil(4 g) was applied uniformly onto porcelain dishes having a diameter of22 cm, and air-dried for one whole day and night.

The above 3 dishes were subjected to a detergency test.

The evaluation method for detergency against starch soil

Remaining starch on the dishes was measured by a photograph to determinethe blue-part-area (P1) generated by iodo-color-reaction. Then, washingrate was obtained from the above P1 and the initial contamination area(So) by the following equation.

    Detergency rate=[(So-P.sub.1)/So]×100

3) Washing composition

    ______________________________________                                        Softanol EP 7045      2 wt. %                                                 (manufactured by Nippon Shokubai                                              Kaguku Industry Co.)                                                          Sodium citrate        20                                                      No. 1 sodium silicate  5                                                      Enzyme                See table 7                                             Sodium Carbonate      balance                                                 ______________________________________                                         (Note) Values for enzymes give activities in the detergent composition.  

4) Results of detergency test

The results are shown in Table 7.

                  TABLE 7                                                         ______________________________________                                                     Samples of  Comparative                                                       present invention                                                                         Compositions                                                      1      2        1       2                                        ______________________________________                                        Enzyme Alkaline    100      250    --    --                                   activity                                                                             pullulanase A                                                          (U/g)  Promozyme   --       --     500   --                                          Termamyl 60 T                                                                             --       --     --    500                                         Detergency   60       70     20    30                                         (%)                                                                    ______________________________________                                         (Notes) Alkaline pullulanse A was obtained in preparation Example 1.          Promozyme is a pullulanase manufactured by Novo Co.                           Termamyl 60 T is an amylase manufactured by Novo Co.                     

Enzyme activities shown in Table 7 were measured by the method below.

As for the 2 kinds of pullulanases, 0.1 ml of enzyme solution was addedto a 0.9 ml substrate solution consisting of 10 mM glycine-NaCl-NaOHbuffer solution (pH 9.0) and pullulan (final concentration in thereaction system was 0.25%) to react at 40° C. for 30 minutes. As forTermamyl 60 T, 0.1 ml of enzyme solution was added to a 0.9 ml ofsubstrate solution consisting of 10 mM glycine-NaCl-NaOH buffer solution(pH 9.0) and soluble starch (final concentration in the reaction systemwas 0.25%) to react at 50° C. for 15 minutes. After the reaction,quantitative determination of the reducing sugar was carried out by themethod of 3,5-dinitrosalicylic acid (DNS). Specifically, 1.0 ml of DNreagent was added to 1.0 ml of reaction mixture, and the mixture washeated at 100° C. for 5 minutes to develop color. After cooling, themixture was diluted with 4.0 ml of deionized water. This solution wassubjected to colorimetric quantitative analysis at wave length of 535nm. One unit (1 U) of enzyme activity was defined to be the amount ofenzyme which released 1 μmol of reducing sugar (as glucose) per minute.

EXAMPLE 2 Detergent for clothes

Washing conditions, detergency tests, and the results were as follows.

1) Artificially contaminated cloths (test material)

Rice flour and boiled rice grains were mixed (9:1), and further mixedwith a mixer after adding an equal amount of tap water. The abovesolution was applied onto a 10 cm×10 cm of a cotton cloth in an amountof 2.5-5% by weight based on the weight of the cloth, then dried at 20°C. for 24 hours.

2) Washing conditions and method

A detergent composition was dissolved into a hard water of 4° DH toprepare a 0.665% detergent solution. Five pieces ofartificially-contaminated cottom cloths were placed in the detergentsolution. After allowing it to stand at 40° C. for one hour, they weretransferred to the stainless beaker to wash at 100 rpm, 20° C., for 10minutes with a Terg-O-Tometer. After rinsing with running water, thecloths were dried at 20° C., for 24 hours, and subjected to quantitativemeasurement.

3) Evaluation of detergency

The weights of the original cloths and the contaminated cloths beforeand after washing were measured. Detergency rate (%) was calculated bythe following equation. ##EQU1##

Values of detergency in Table 8 are averages of 5 pieces.

4) Detergent composition

    ______________________________________                                        Sodium linear dodecylbenzene sulfonate                                                               15      wt. %                                          Sodium alkylethoxy sulfate                                                                           5                                                      (C.sub.14 -C.sub.15, EO = 3 mol)                                              4A-type zeolite        15                                                     Sodium silicate        15                                                     Sodium carbonate       15                                                     Sodium polyacrylate (MW = 8000)                                                                      1.5                                                    Polyethylene glycol (MW = 6000)                                                                      1.5                                                    Enzyme                 Table 8                                                Fluorescent dye        0.5                                                    Glauber's salt         balance                                                Water                  5                                                      ______________________________________                                         (notes) Values for enzymes give activities in the detergent composition. 

5) Results of detergency tests

The results are shown in Table 8.

                  TABLE 8                                                         ______________________________________                                                     Samples of the                                                                            Comparative                                                       present invention                                                                         Compositions                                                      3      4        3       4                                        ______________________________________                                        Enzyme Alkaline    100      250    --    --                                   activity                                                                             pullulanase A                                                          (U/g)  Promozyme   --       --     500   --                                          Termamyl 60 T                                                                             --       --     --    500                                         Detergency   50       70     25    40                                         (%)                                                                    ______________________________________                                    

The measurement of enzyme activities in Table 8 was the same asexplained the data in Table 7.

EXAMPLE 3 Detergent composition for an automatic dishwasher

Detergency test was carried out in the same manner as in Example 1except for using alkaline pullulanase B as an enzyme.

The results are shown in Tables 9 and 10.

                  TABLE 9                                                         ______________________________________                                                      Samples of the                                                                            Comparative                                                       present invention                                                                         Compositions                                                      5      6        5                                               ______________________________________                                        Enzyme Alkaline      7       14     --                                        activity                                                                             pullulanase B                                                          (U/g)  Termamyl 60 T                                                                              --       --     14                                               Detergency   60       80     50                                               (%)                                                                    ______________________________________                                         (Notes) Alkaline pullulanse B was obtained in Preparation Example 2.          Termamyl 60 T is an amylase manufactured by Novo Co.                     

Enzyme activities shown in Table 9 were measured by the method of"Amylase B-test Wako" (CM-amylose DEX method). 1U was defined to be anenzyme unit where 100 ml of enzyme decomposes the right amounts of 10 mgof starch.

                  TABLE 10                                                        ______________________________________                                                      Samples of the                                                                            Comparative                                                       present invention                                                                         Compositions                                                      7      8        6                                               ______________________________________                                        Enzyme Alkaline     50       80     --                                        activity                                                                             pullulanase B                                                          (U/g)  Promozyme    --       --     200                                              Detergency   60       80      30                                              (%)                                                                    ______________________________________                                         (Notes) Alkaline pullulanse B was obtained in Preparation Example 2.          Promozyme is a pullulanase manufactured by Novo Co.                      

Enzyme activities shown in Table 10 were measured by the followingmethod.

A 0.1 ml enzyme solution was added to 0.9 ml of a substrate solutionconsisting of 10 mM glycine-NaCl-NaOH buffer solution (pH 9.0) andpullulan (final concentration in the reaction system was 0.25%) whichwas to react at 40° C. for 30 minutes. After the reaction, quantitativedetermination of reducing sugar was carried out by the method of3,5-dinitro salicylic acid (DNS). In detail, 1.0 ml of DNS reagent wasadded to 1.0 ml of the reaction mixture, and the mixture was heated at100° C. for 5 minutes to develop color. After cooling, the mixture wasdiluted with 4.0 ml of deionized water. This solution was subjected tocolorimetric quantitative analysis at wave length of 535 nm.

One unit (1U) of enzyme activity was defined to be the amount of enzymewhich released 1 μmol of reducing sugar (as glucose) per minute.

EXAMPLE 4 Detergent composition for clothes

Detergents of the following compositions were prepared. Detergency testwas carried out in the same manner as in Example 2.

1) Detergent composition

    ______________________________________                                        Sodium linear dodecylbenzene sulfonate                                                               15      wt. %                                          Sodium alkylethoxy sulfate                                                                           5                                                      (C.sub.14 -C.sub.15, EO = 3 mol)                                              4A type zeolite        15                                                     Sodium silicate        15                                                     Sodium carbonate       15                                                     Sodium polyacrylate (MW = 8000)                                                                      1.5                                                    Polyethyleglycol (MW = 6000)                                                                         1.5                                                    Enzyme                 Table 11 or                                                                   Table 12                                               Fluorescent dye        0.5                                                    Glauber's salt         balance                                                Water                  5                                                      ______________________________________                                         (notes) Values for enzymes mean activities in the detergent composition. 

2) Results of detergency test

The results are shown in Tables 11 and 12.

                  TABLE 11                                                        ______________________________________                                                      Samples of the                                                                            Comparative                                                       present invention                                                                         Compositions                                                      9      10       7                                               ______________________________________                                        Enzyme Alkaline      8       14     --                                        activity                                                                             pullulanase B                                                          (U/g)  Termamyl 60 T                                                                              --       --     14                                               Detergency   65       80     50                                               (%)                                                                    ______________________________________                                         (Notes) Alkaline pullulanse B was obtained in Preparation Example 2.          Termamyl 60 T is an amylase manufactured by Novo Co.                     

Enzyme activities shown in Table 11 were measured by the methodexplained for Table 9 data.

                  TABLE 12                                                        ______________________________________                                                      Samples of the                                                                            Comparative                                                       present invention                                                                         Compositions                                                      11     12       8                                               ______________________________________                                        Enzyme Alkaline     50       80     --                                        activity                                                                             pullulanase B                                                          (U/g)  Promozyme    --       --     250                                              Detergency   55       80      35                                              (%)                                                                    ______________________________________                                         (Notes) Alkaline pullulanse B was obtained in Preparation Example 2.          Promozyme is a pullulanase manufactured by Novo Co.                      

Enzyme activities shown in Table 12 were measured by the methodexplained for Table 10 data.

As explained above, the detergent compositions of the present inventionexhibited an excellent detergency against starch soils within a normaloperation time.

EXAMPLE 5 Detergent composition for automatic dish washer

The following detergent composition was prepared.

    ______________________________________                                        (Composition)                                                                 Softanol EP-7085 .sup.1)                                                                          4.5 wt. %                                                 Trisodium citrate   20.0                                                      Sokalan CP-5 powder .sup.2)                                                                       5.0                                                       Lipase .sup.3)      1.0                                                       Alkaline Pullulanase A .sup.4)                                                                    250 .sup.4)                                               Sodium hydrogencarbonate                                                                          50.0                                                      Sodium sulfate      balance                                                   ______________________________________                                         Note)                                                                         .sup.1) Softanol EP7085: Addition product of secalcohol added with 7 mols     of ethylene oxide and 8.5 mols of propylene oxide manufactured by Nippon      Shokubai Kagaku Kogyo K.K.                                                    .sup.2) Sokalan CP5 powder: Salt of copolymer of acrylic acid and maleic      anhydride, MW = about 70,000 (manufactured by BASF)                           .sup.3) Lipase AKG: Manufactured by Amano Seiyaku K.K.                        .sup.4) Alkaline pullulanase: Obtained in Preparation Example 1. The give     value indicates the activity (Unit/g detergent) of one gram of detergent.

EXAMPLE 6 Detergent composition for automatic dish washer

The following detergent composition was prepared.

    ______________________________________                                        (Composition)                                                                 Pluronic L92 .sup.5)                                                                              4 wt. %                                                   Trisodium citrate   30                                                        Lipase .sup.6)      1.5                                                       Alkaline pullulanase B .sup.7)                                                                    80 .sup.7)                                                Sodium hydrogencarbonate                                                                          30                                                        Sodium metasilicate 2                                                         Sodium sulfate      balance                                                   ______________________________________                                         Note)                                                                         .sup.5) Pluronic L92: Polyoxyethylenepolyoxypropylene block copolymer,        average molecular weight = 3,500 (manufactured by Asahi Denka K.K.)           .sup.6) Lipase 30T: Manufactured by Novo Industry Co.                         .sup.7) Obtained in Preparation Example 2. The given value indicates the      activity (Unit/g detergent) of one gram of detergent.                    

The detergent compositions of Examples 5 and 6 exhibited excellentdetergency not only against starch soils, but also against complexstains of starch soil, fats and oils protein soils.

Obviously, numerous modifications and variations of the presentinvention are possible in light of the above teachings. It is thereforeto be understood that within the scope of the appended claims thepresent invention may be practiced otherwise than as specificallydescribed herein.

What is claimed is:
 1. A detergent composition comprising 0.1-10 wt. %alkaline pullulanase B and 0.5-60 wt. % surfactant, wherein saidalkaline pullulanase B has the following enzymologicalcharacteristics:1) action acts on pullulan and soluble starch to producemainly maltotriose from pullulan and mainly maltotetraose andmaltopentaose from soluble starch, and also acts on glycogen to producemaltotetraose and maltopentaose; 2) substrate specificity acts onpullulan, soluble starch, and glycogen; 3) working pH and optimum pHrange the working pH on pullulan being in the range of 5-12 with anoptimum pH in the range of 8.5-10, and the working pH on soluble starchbeing in the range of 4-12 with an optimum pH in the range of 7-9.5; 4)pH stability stable in the pH range of 6-10.5 against pullulan, and inthe pH range of 4-12 against soluble starch (treatment conditions: 45°C., 10 minutes); 5) working temperatures and optimum temperature Acts onpullulan and soluble starch at the temperature range of 10°-65° C. withan optimum temperature being about 50° C.; 6) thermal stability quitestable up to 45° C. (treatment condition: in 10 mM glycine-NaCl-NaOHbuffer solution, at pH 9.5 for 30 minutes); 7) Molecular weight 200.000±5,000 daltons as determined by sodium dodecylsulfate (SDS)electrophoresis;8) effects of metal ions pullulanase activities beingadversely effected by Hg²⁺, Mn²⁺ and Pb²⁺, and α-amylase activities areadversely affected by Hg²⁺, Mn²⁺, Pb²⁺, Zn²⁺ and Cd²⁺ ; 9) effects ofsurfactants activities being scarcely adversely affected by surfactantssuch as linear alkylbenzene sulfonate, sodium polyoxyethylene alkylsulfate, sodium α-olefin sulfonate, sodium α-sulfonated fatty acidester, sodium alkyl sulfonate, sodium dodecyl sulfate, soaps andSoftanol (trade-mark), said alkaline pullulanase B being produced by aprocess comprising culturing a microorganism producing an alkalinepullulanase which is named as Bacillus sp. KSM-AP 1378 deposited as FERMBP-3048, and isolating the alkaline pullulanase B from the culturedmedium.
 2. The detergent composition of claim 1 which furthercontains:0-90 wt. % alkaline agents and/or inorganic electrolytes, 0-50wt. % divalent metal ion scavengers, and 0-85 wt. % bleaching agents.